Co-Crystals and Pharmaceutical Compositions Comprising the same

ABSTRACT

The invention relates to co-crystals and compositions each comprising VX-950 and a co-crystal former selected from the group consisting of 3-methoxy-4-hydroxybenzoic acid, 2,4-dihydroxybenzoic acid, and 2,5-dihydroxybenzoic acid. Also within the scope of this invention are methods of making and using the same.

CROSS REFERENCE

This application claims priority to U.S. Application No. 60/969,023,filed on Aug. 30, 2007, the content of which is incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

Infection by hepatitis C virus (“HCV”) is a compelling human medicalproblem. HCV is recognized as the causative agent for most cases ofnon-A, non-B hepatitis, with an estimated human prevalence of 3%globally [A. Alberti et al., “Natural History of Hepatitis C,” J.Hepatology, 31 (Suppl. 1), pp. 17-24 (1999)]. Nearly four millionindividuals may be infected in the United States alone [M. J. Alter etal., “The Epidemiology of Viral Hepatitis in the United States,Gastroenterol. Clin. North Am., 23, pp. 437-455 (1994); M. J. Alter“Hepatitis C Virus Infection in the United States,” J. Hepatology, 31(Suppl. 1), pp. 88-91 (1999)].

Upon first exposure to HCV, only about 20% of infected individualsdevelop acute clinical hepatitis while others appear to resolve theinfection spontaneously. In almost 70% of instances, however, the virusestablishes a chronic infection that persists for decades [S. Iwarson,“The Natural Course of Chronic Hepatitis,” FEMS Microbiology Reviews,14, pp. 201-204 (1994); D. Lavanchy, “Global Surveillance and Control ofHepatitis C,” J. Viral Hepatitis, 6, pp. 35-47 (1999)]. This usuallyresults in recurrent and progressively worsening liver inflammation,which often leads to more severe disease states such as cirrhosis andhepatocellular carcinoma [M. C. Kew, “Hepatitis C and HepatocellularCarcinoma”, FEMS Microbiology Reviews, 14, pp. 211-220 (1994); I. Saitoet al., “Hepatitis C Virus Infection is Associated with the Developmentof Hepatocellular Carcinoma,” Proc. Natl. Acad. Sci. USA, 87, pp.6547-6549 (1990)]. Unfortunately, there are no broadly effectivetreatments for the debilitating progression of chronic HCV.

The HCV genome encodes a polyprotein of 3010-3033 amino acids [Q. L.Choo, et al., “Genetic Organization and Diversity of the Hepatitis CVirus,” Proc. Natl. Acad. Sci. USA, 88, pp. 2451-2455 (1991); N. Kato etal., “Molecular Cloning of the Human Hepatitis C Virus Genome FromJapanese Patients with Non-A, Non-B Hepatitis,” Proc. Natl. Acad. Sci.USA, 87, pp. 9524-9528 (1990); A. Takamizawa et al., “Structure andOrganization of the Hepatitis C Virus Genome Isolated From HumanCarriers,” J. Virol., 65, pp. 1105-1113 (1991)]. The HCV nonstructural(NS) proteins are presumed to provide the essential catalytic machineryfor viral replication. The NS proteins are derived by proteolyticcleavage of the polyprotein [R. Bartenschlager et al., “NonstructuralProtein 3 of the Hepatitis C Virus Encodes a Serine-Type ProteinaseRequired for Cleavage at the NS3/4 and NS4/5 Junctions,” J. Virol., 67,pp. 3835-3844 (1993); A. Grakoui et al., “Characterization of theHepatitis C Virus-Encoded Serine Proteinase: Determination ofProteinase-Dependent Polyprotein Cleavage Sites,” J. Virol., 67, pp.2832-2843 (1993); A. Grakoui et al., “Expression and Identification ofHepatitis C Virus Polyprotein Cleavage Products,” J. Virol., 67, pp.1385-1395 (1993); L. Tomei et al., “NS3 is a serine protease requiredfor processing of hepatitis C virus polyprotein”, J. Virol., 67, pp.4017-4026 (1993)].

The HCV NS protein 3 (NS3) is essential for viral replication andinfectivity [Kolykhalov, J. Virology, Volume 74, pp. 2046-2051 2000“Mutations at the HCV NS3 Serine Protease Catalytic Triad abolishinfectivity of HCV RNA in Chimpanzees]. It is known that mutations inthe yellow fever virus NS3 protease decrease viral infectivity[Chambers, T. J. et al., “Evidence that the N-terminal Domain ofNonstructural Protein NS3 From Yellow Fever Virus is a Serine ProteaseResponsible for Site-Specific Cleavages in the Viral Polyprotein”, Proc.Natl. Acad. Sci. USA, 87, pp. 8898-8902 (1990)]. The first 181 aminoacids of NS3 (residues 1027-1207 of the viral polyprotein) have beenshown to contain the serine protease domain of NS3 that processes allfour downstream sites of the HCV polyprotein [C. Lin et al., “HepatitisC Virus NS3 Serine Proteinase: Trans-Cleavage Requirements andProcessing Kinetics”, J. Virol., 68, pp. 8147-8157 (1994)].

The HCV NS3 serine protease and its associated cofactor, NS4A, helpprocess all of the viral enzymes, and are thus considered essential forviral replication. his processing appears to be analogous to thatcarried out by the human immunodeficiency virus aspartyl protease, whichis also involved in viral enzyme processing. HIV protease inhibitors,which inhibit viral protein processing, are potent antiviral agents inman indicating that interrupting this stage of the viral life cycleresults in therapeutically active agents. Consequently, HCV NS3 serineprotease is also an attractive target for drug discovery.

Until recently, the only established therapy for HCV disease wasinterferon treatment. However, interferons have significant side effects[M. A. Wlaker et al., “Hepatitis C Virus: An Overview of CurrentApproaches and Progress,” DDT, 4, pp. 518-29 (1999); D. Moradpour etal., “Current and Evolving Therapies for Hepatitis C,” Eur. J.Gastroenterol. Hepatol., 11, pp. 1199-1202 (1999); H. L. A. Janssen etal. “Suicide Associated with Alfa-Interferon Therapy for Chronic ViralHepatitis,” J. Hepatol., 21, pp. 241-243 (1994); P. F. Renault et al.,“Side Effects of Alpha Interferon,” Seminars in Liver Disease, 9, pp.273-277 (1989)] and induce long term remission in only a fraction (˜25%)of cases [O. Weiland, “Interferon Therapy in Chronic Hepatitis C VirusInfection”, FEMS Microbiol. Rev., 14, pp. 279-288 (1994)]. Recentintroductions of the pegylated forms of interferon (PEG-INTRON® andPEGASYS®) and the combination therapy of ribavirin and interferon(REBETROL®) have resulted in only modest improvements in remission ratesand only partial reductions in side effects. Moreover, the prospects foreffective anti-HCV vaccines remain uncertain.

Thus, there is a need for more effective anti-HCV therapies. Suchinhibitors would have therapeutic potential as protease inhibitors,particularly as serine protease inhibitors, and more particularly as HCVNS3 protease inhibitors. Specifically, such compounds may be useful asantiviral agents, particularly as anti-HCV agents.

VX-950, an HCV inhibitor with its structure shown below is such acompound in need. VX-950 is described in PCT Publication Number WO02/18369, which is incorporated herein by reference in its entirety.

SUMMARY OF THE INVENTION

In general, the present invention relates to co-crystals comprising theHCV inhibitor VX-950 and a co-crystal former, as well as compositionscontaining the VX-950 co-crystals. A co-crystal former can be apharmacologically inert excipient that alters the crystal form of asolid drug through the formation of co-crystals, clathrates, or othercrystalline solid forms. It is within the meaning of “co-form” usedherein. Under certain circumstances, VX-950 and the co-crystal formertogether may form a multi-component single phase crystalline solid orcomposition, i.e., a co-crystal. Compared to their free forms, specificVX-950 co-crystals are advantageous as they may possess improveddissolution, higher aqueous solubility, or greater solid state physicalstability than amorphous VX-950 dispersions. The specific VX-950co-crystals provide a reduced mass of the dosage form and thereforelower pill burden since the VX-950 co-crystals also exhibit higher bulkdensities relative to amorphous forms. Further, VX-950 co-crystalsprovide manufacturing advantages relative to amorphous forms whichrequire spray drying, melt extrusion, lyophilization, or precipitation.

In one aspect, the VX-950 co-crystals provided by this invention containVX-950 and 2,4-dihydroxybenzoic acid as a co-crystal former compound. Insome embodiments, the molar ratio of VX-950 and 2,4-dihydroxybenzoicacid in the co-crystals is in the range of about 5:1 to about 1:5 (e.g.,about 1:1). In some embodiments, the co-crystal has at least two of thefour X-ray powder diffraction peaks at about 7.6, 8.5, 9.6, and 11.9degree 2-Theta, each with a standard deviation of about +/−0.3° 2-Theta.In some embodiments, the co-crystal has a peak in its DSC thermogram atabout 150.81° C., with a standard deviation of about +/−5° C.

In another aspect, the VX-950 co-crystals provided by this inventioncontain VX-950 and 2,5-dihydroxybenzoic acid as a co-crystal formercompound. In some embodiments, the molar ratio of VX-950 and2,5-dihydroxybenzoic acid in the co-crystals is in the range of about5:1 to about 1:5 (e.g., about 1:1). In some embodiments, the co-crystalseach have at least two of the four X-ray powder diffraction peaks atabout 8.3, 9.6, 11.7, and 17.1 degree 2-Theta, each with a standarddeviation of about +/−0.3 degree 2-Theta (i.e., ° 2-Theta). In someother embodiments, the co-crystals have a peak in its DSC thermogram atabout 163.48° C. with a standard deviation of about +/−5° C.

In still another aspect, the present invention provides co-crystalscomprising VX-950 and 3-methoxy-4-hydroxybenzoic acid as a co-crystalformer compound. In some embodiments, the molar ratio of VX-950 and3-methoxy-4-hydroxybenzoic acid in the co-crystals is in the range ofabout 5:1 to about 1:5 (e.g., about 1:1). In some embodiments, theco-crystals each have at least two of the four X-ray powder diffractionpeaks at about 7.8, 8.6, 11.4, and 11.9 degree 2-Theta, each with astandard deviation of about +/−0.3 degree 2-Theta. In some embodiments,the co-crystals each have a peak in its DSC thermogram at about 178.09°C. with a standard deviation of about +/−5° C.

In another aspect, the invention provides compositions each containingVX-950; a co-crystal former selected from the group consisting of2,4-dihydroxybenzoic acid, 2,5-dihydroxybenzoic acid, and3-methoxy-4-hydroxybenzoic acid; and a solvent selected from the groupconsisting of acetonitrile, dichloromethane, ethyl acetate, ethanol,acetone, or a mixture thereof. In some embodiments, VX-950, theco-crystal former, and the solvent together may take the crystallineform (i.e., forming a co-crystal). Due to the presence of the solvent,the co-crystal may be a solvate. In some other embodiments, the solventis a mixture of acetonitrile and dichloromethane (e.g., of about 1:1ratio by volume). In some embodiments, the molar ratio of VX-950 and4-amino salicylic acid is in the range of about 5:1 to about 1:5 (e.g.,about 1:1). In some embodiments, the molar ratio of VX-950 and thesolvent is in the range of about 1:0.05 to about 1:1 (e.g., about 1:0.34or about 1:0.5).

These compositions may have applications, among others, in treatingdiseases implicated by or associated with HCV. As such, also within thescope of the invention are pharmaceutical compositions each containingVX-950 and a co-crystal former identified above, in an appropriate molarratio. The pharmaceutical composition may optionally contain a solvent(e.g., acetonitrile, dichloromethane, ethyl acetate, ethanol, oracetone) for forming a solvate. Additionally, the pharmaceuticalcompositions may further contain a pharmaceutically acceptable diluent,solvent, excipient, carrier, or solubilizing agent.

Still also within the scope of this invention is a method for making aco-crystal described above. The method may include the steps of (a)providing VX-950, (b) providing a co-crystal former selected from thegroup consisting of 2,4-dihydroxybenzoic acid, 2,5-dihydroxybenzoicacid, and 3-methoxy-4-hydroxybenzoic acid (optionally in a solvent,e.g., ethanol, acetonitrile, dichloromethane, or a mixture thereof, forforming a solvate), (c) grinding, heating, co-subliming, co-melting, orcontacting in solution VX-950 with the co-crystal former undercrystallization condition so as to form the co-crystal in solid phase,and (d) optionally isolating the co-crystal formed by step (c).

Still within the scope of this invention is a method for modulating achemical or physical of interest of a co-crystal described above. Themethod may include the steps of (a) measuring the chemical or physicalproperty of interest for VX-950 and a co-crystal former selected fromthe group consisting of 2,4-dihydroxybenzoic acid, 2,5-dihydroxybenzoicacid, and 3-methoxy-4-hydroxybenzoic acid, (b) determining the molefraction of the VX-950 and the co-crystal former that will result in thedesired modulation of the chemical or physical property of interest, and(c) preparing the co-crystal with the molar fraction determined in step(b).

The compositions and co-crystals of this invention can be used fortreating diseases implicated by or associated with HCV. Thus, alsowithin the scope of this invention is a method of treating suchdiseases, which comprising administering to a subject in need thereof atherapeutically effective amount of a co-crystal of this invention or acomposition of this invention.

The compositions and co-crystals of this invention can also be used asseeds to prepare additional co-crystals containing an active ingredientthat can be the same as or different from VX-950, and a co-crystalformer that can also be the same as or different from the groupconsisting of 2,4-dihydroxybenzoic acid, 2,5-dihydroxybenzoic acid, and3-methoxy-4-hydroxybenzoic acid. For instance, a small amount of aco-crystal of this invention can be placed into a solution containingthe desired active ingredient and the co-crystal former and the mixtureis allowed to sit so that additional co-crystal can be formed with andgrown out of the existing co-crystal.

Additionally, the compositions and co-crystals of this invention can beused as research tools. For instance, they can be used to study thepharmacological properties (such as bioavailability, metabolism, andefficacy) of VX-950 in different form and under condition, or to developvarious VX-950 formulations for best delivery and absorption.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows XRPD of the co-crystal of VX-950 and 2,5-dihydroxybenzoicacid.

FIG. 2 shows a TGA spectrum of the co-crystal of VX-950 and2,5-dihydroxybenzoic acid.

FIG. 3 shows a DSC spectrum of the co-crystal of VX-950 and2,5-dihydroxybenzoic acid.

FIG. 4 shows XRPD of the co-crystal of VX-950 and3-methoxy-4-hydroxybenzoic acid.

FIG. 5 shows a TGA spectrum of the co-crystal of VX-950 and3-methoxy-4-hydroxybenzoic acid.

FIG. 6 shows a DSC spectrum of the co-crystal of VX-950 and3-methoxy-4-hydroxybenzoic acid.

FIG. 7 shows XRPD of the co-crystal of VX-950 and 2,4-dihydroxybenzoicacid.

FIG. 8 shows a TGA spectrum of the co-crystal of VX-950 and2,4-dihydroxybenzoic acid.

FIG. 9 shows a DSC spectrum of the co-crystal of VX-950 and2,4-dihydroxybenzoic acid.

DETAILED DESCRIPTION OF THE INVENTION

Methods for preparing and characterizing a co-crystal are welldocumented in the literature. See, e.g., Trask et al., Chem. Commun.,2004, 890-891; and O. Almarsson and M. J. Zaworotko, Chem. Commun.,2004, 1889-1896. These methods in general are also suitable forpreparing and characterizing co-crystals of this invention.

Additionally, the following specific methods can be used to identifyco-crystal formers suitable for making co-crystals, particularly theco-crystals of this invention.

Initial identification of or screening for a possible co-crystal formerfor VX-950 can be conducted on a scale of a few milligrams in a 96-wellplate. Visual comparison of the XRPD results to the known crystallineVX-950 diffraction pattern can be used as a screen for new crystal formsand/or altered crystal lattice dimensions that could indicateincorporation of a co-crystal former into a crystal. Oxalic acid,4-amino salicylic acid, and salicylic acid, etc. have been identified bythis initial screening as possible candidates for forming co-crystalswith VX-950.

The results of the initial screening can be used for modeling work toidentify additional co-crystal formers for VX-950. For instance, due tobetter physical and chemical properties, 4-amino salicylic acid can beused as a lead molecule in identifying other possible co-crystal formersfor VX-950 via molecular modeling. Specifically, a model of 4-ASA can bebuilt using the Quanta software package (Accelrys Inc., San Diego,Calif.) and be complexed with the structure of a single molecule ofVX-950 obtained by single crystal x-ray diffraction. The 4-ASA moleculecan be placed manually at different positions around VX-950 to form themaximum number of hydrogen bonds between the two molecules. Thepositional coordinates of the 4-ASA molecule are energy-minimized whilethe VX-950 molecule is held fixed. An adopted-basis Newton-Raphsonmethod available in Quanta can be used for energy minimization usingdefault settings and a distance-dependent dielectric. AutoNom software(MDL Information Systems, GmbH) can be used to convert the names ofchemical compounds in the FDA's EAFUS (Everything Added to Food, US) andGRAS (Generally Regarded As Safe) lists into 2D structures in SMILESformat to produce a database of structures. The database can then besearched for new co-crystal formers that fit the pharmacophoreidentified with 4-ASA. Acceptable pharmacophores have local energyminima similar to that of VX-950 and 4-ASA.

DSC can also be used for screening co-crystal formers. In screening byDSC, physical mixtures of co-crystal formers with VX-950 that showedevidence of solid-phase interactions during DSC (i.e., the formation ofeutectic melts) are probably more likely to form co-crystals. To detectan interaction between VX-950 and the co-crystal former, the componentscan be blended in a 1:1 molar ratio and subjected to a DSC temperatureramp method from the room temperature to, e.g., 300° C. in 10° C.increments. Blends showing a new thermal-event (i.e., an endotherm) thatdiffers in temperature from endotherms of the pure components areselected. When the new thermal transition is observed in addition tothat of the one of original components, the molar ratio between VX-950and the co-crystal former can then be adjusted in an attempt to yieldthe new thermal transition only. The observed transition temperaturescan be plotted as a function of composition to produce phase diagramsfor the binary mixtures. Combinations of VX-950 and co-crystal formerthat produce new thermal transitions on DSC can then be scaled up toproduce larger quantities (e.g., grams) as described above.

Mixtures of VX-950 and co-crystal formers with new thermal transitionscan be produced in large quantity (i.e., scaled-up), e.g., by using theball-milling, solvent-evaporation, melting with and without solvents,slurry conversion, blending, sublimation, or modeling. Some of thesemethods are described in detail below. The products thus prepared can beanalyzed or characterized by such known methods as XRPD, TGA, and DSC,and their solubility and stability in an aqueous medium can also bemeasured by methods known in the art.

Ball-milling: Equal molar amounts of VX-950 and a co-crystal former aremixed with an appropriate solvent. The mixture is then milled using aball-mill apparatus, e.g., Retsch MM200 (GlenMills Inc., Clifton, N.J.)for 3 hours at a frequency of 15 Hz. The mixture is then placed in themilling compartment made of sintered corundum. After milling, thematerial is placed in a screw cap scintillation vials (uncapped) anddried under vacuum at the room temperature. XRPD and DSC analyses can beperformed to characterize the resulting mixture.

Melting in a Reaction Block: Equi-molar amounts of VX-950 and theco-crystal former are mixed, with or without a solvent. The mixture isthen placed in a reaction block, e.g., Model RR98072 by RadleysDiscovery Technologies (Essex, UK) with the lid closed and heated to thetemperature identified by DSC of the new thermal transition. The mixtureis then held for a period of time at the transition temperature beforethe reaction block is opened and the resulting mixture cooled underambient conditions.

Solvent Evaporation: VX-950 and a potential co-crystal former aredissolved separately into a volatile solvent or a solvent mixture (e.g.,50:50 toluene:acetonitrile). Dissolution can be aided by agitation andsonication until a clear solution is obtained. The VX-950 solution isthen mixed with the co-crystal former solution in screw-capscintillation vials at the desired molar ratio. The vials are placeduncapped under reduced pressure and solvent allowed to evaporate todryness, typically over a period of several days. Solid (crystalline)material is obtained and analyzed.

As mentioned above, co-crystals of this invention can be analyzed bymethods known in the art for characterizing solid or crystallinematerials. Examples of characterization methods includethermogravimetric analysis (TGA), differential scanning calorimetry(DSC), X-ray powder diffraction (XRPD), solubility analyses, dynamicvapor sorption, infrared off-gas analysis, and suspension stability. TGAcan be used to investigate the presence of residual solvents in aco-crystal sample, and to identify the temperature at whichdecomposition of each co-crystal sample occurs. DSC can be used to lookfor thermo-transitions occurring in a co-crystal sample as a function oftemperature and determine the melting point of each co-crystal sample.XRPD can be used for structural characterization of the co-crystal.Solubility analysis can be performed to reflect the changes in thephysical state of each co-crystal sample. And suspension stabilityanalysis can be used to determine the chemical stability of a co-crystalsample in a solvent. Described in great detail are some of such methods.

X-ray Powder Diffraction (XRPD): XRPD can be used to characterize thephysical form of the material by recording its original pattern andmonitoring changes in the pattern with time. The XRPD pattern can beobtained at the room temperature in reflection mode, e.g., by using aBruker D8 Discover diffractometer that is equipped with a sealed tubesource and a Hi-Star area detector (Bruker AXS, Madison, Wis., USA). Acopper target X-ray tube (Siemens) can be operated, e.g., at 40 kV and35 mA. Graphite monochromator and 0.5 mm collimator provided by Brukercan be used to produce parallel, monochromatic beam (CuKa, λ=1.5418 Å).The distance between the sample and the detector can be approximately 30cm. The sample can be placed on a Si zero-background wafer (e.g., fromThe Gem Dugout, State College, Pa.) which is then positioned andcentered on XYZ platform. Data can bee acquired using GADDS software forWindows NT, version 4.1.16 (Bruker AXS, Madison, Wis., USA). Two framescan be registered with an exposure time of 120 seconds per frame each at2 different 2θ angles: 8° and 26° 2-Theta. The sample is oscillated inboth X and Y directions with an amplitude of 1 mm during the exposure.The data can then be subsequently integrated over the range of 3° to 41°2-Theta with a step size of 0.02° 2-Theta and merged into one continuouspattern. Corundum plate (NIST standard 1976) can be used to calibratethe instrument.

Differential Scanning Calorimetry (DSC): DSC can be used to detectthermal transitions occurring in the sample as a function of temperatureand to determine the melting point of crystalline materials. It isperformed, e.g., using an MDSC Q100 differential scanning calorimeter(TA Instruments, New Castle, Del.) calibrated with indium. The samplescan be prepared in aluminum pans crimped with a single pinhole with thesample size being, e.g., approximately 2 mg. Each run is initiallyequilibrated to 25° C. followed by a ramp of 10° C./minute to 300° C.VX-950 degrades upon melting, degradation onset is about 240° C. Thedata can be collected by Thermal Advantage Q Series™ software andanalyzed by Universal Analysis software (TA Instruments, New Castle,Del.).

Thermogravimetric Analysis (TGA): TGA can be used to investigate thepresence of residual solvents in the samples, and to identify thetemperature at which decomposition of the sample occurs. For instance, aModel Q500 Thermogravimetric Analyzer (TA Instruments, New Castle, Del.)can be used for TGA measurement. The sample can weigh in the range ofabout 3-8 mg, and be heated at the rate of about 10° C./minute to afinal temperature of, e.g., 300° C. The data can be, e.g., collected byThermal Advantage Q Series™ software and analyzed by Universal Analysissoftware (TA Instruments, New Castle, Del.).

Fourier Transform Infrared (FT-IR) Spectrometry: FT-IR can be used toinvestigate hydrogen bonding in blends of VX-950 with a co-crystalformer at different molar ratios. Infrared transmission spectra can beobtained, e.g., from KBr pellets with Nexus 670 spectrometer (ThermoElectron Corp.; Madison, Wis.) from 4000 to 625 cm⁻¹.

Solubility Determination: Solubility can be expressed in VX-950equivalents. It can be measured to reflect the changes in the physicalstate of the material, and to monitor progress toward the goal ofenhancing VX-950 solubility. Specifically, an aliquot of the materialcan be placed in an aqueous medium with a target solubility of 10 mg/mL.At set time points, aliquots of supernatant are withdrawn, filteredthrough a 0.45 micron filter (e.g., Millex; Millipore, Billerica, Mass.)and analyzed using HPLC (e.g., Agilent 1100; Palo Alto, Calif.). Thesamples are run isocratically with the detector, e.g., set at 270 nm anda flow rate of 1 mL/min on an XTerra® Phenyl column 150 mm×4.6 mm, 3.5μm particle size (P/N 186001144) (Waters Corp., Milford, Mass.). Themobile phase contained potassium phosphate buffer (10 mM, pH=7.0) andmethanol in a 60:40 (v/v) ratio. The concentrations of VX-950 can bedetermined by comparing chromatographic peak areas with a calibrationcurve produced using standards of known concentration.

Hotstage Microscopy: Microscope images can be taken, e.g., with anOlympus BX51 confocal microscope with polarized films, an SLMPlan 50×infinity corrected objective, a C-5050 digital camera, and an Instechotstage with a variable temperature controller. The experimentalprocedure consists of a linear heating ramp between differenttemperature steps in which the samples are allowed to equilibrate forseveral minutes. Digital images are collected manually throughout theramp to capture any transitions that occurred.

An effective amount of co-crystals or compositions of this invention,each including VX-950 and a co-crystal former (e.g., 4-hydroxybenzoicacid, 4-amino salicylic acid (with acetonitrile), phenyl alanine,threonine, adipic acid, succinic acetate, praline, methyl4-hydroxybenzoate, anthranilic acid, d-Biotin, or tartaric acid) can beused to treat diseases implicated or associated with the HCV. Aneffective amount is the amount which is required to confer a therapeuticeffect on the treated subject, e.g. a patient. The effective amount of aco-crystal of VX-950 and the co-crystal former is between about 0.1mg/kg to about 150 mg/kg (e.g., from about 1 mg/kg to about 60 mg/kg).Effective doses will also vary, as recognized by those skilled in theart, dependent on route of administration, excipient usage, and thepossibility of co-usage with other therapeutic treatments including useof other therapeutic agents and/or therapy.

The co-crystals or pharmaceutical compositions of this invention can beadministered to the subject in need thereof (e.g., cells, a tissue, or apatient (including an animal or a human)) by any method that permits thedelivery of the compound VX-950, e.g., orally, intravenously, orparenterally. For instance, they can be administered via pills, tablets,capsules, aerosols, suppositories, liquid formulations for ingestion orinjection or for use as eye or ear drops, dietary supplements, andtopical preparations.

The pharmaceutical compositions can include diluents, solvents,excipients and carriers such as water, Ringer's solution, isotonicsaline, 5% glucose, and isotonic sodium chloride solution. In anotherembodiment, the pharmaceutical composition can further include asolubilizing agent such as cyclodextrin. Additional examples of suitablediluents, solvents, excipients, carriers, and solubilizing agents can befound, e.g., in U.S. Pharmacopeia 23/National Formulary 18, Rockville,Md., U.S. Pharmacopeia Convention, Inc., (1995); Ansel H C, Popovich NG, Allen Jr L V. Pharmaceutical Dosage Forms and Drug Delivery Systems,Baltimore Md., Williams &Wilkins, (1995); Gennaro A R., Remingtons: TheScience and Practice of Pharmacy, Easton Pa., Mack Publishing Co.,(1995); Wade, A., Weller, P. J., Handbook of Pharmaceutical Excipients,2nd Ed, Washington D.C., American Pharmaceutical Association, (1994);Baner G S, Rhodes C T. Modern Pharmaceutics, 3rd Ed., New York, MarcelDekker, Inc., (1995); Ranade V V, Hollinger Mass., Drug DeliverySystems, Boca Raton, CRC Press, (1996).

The pharmaceutical compositions can also include aqueous solutions ofthe co-crystal, in an isotonic saline, 5% glucose or other well-knownpharmaceutically acceptable excipient(s). Solubilizing agents such ascyclodextrins, or other solubilizing agents well-known to those familiarwith the art, can be utilized as pharmaceutical excipients for deliveryof the therapeutic compound VX-950. As to route of administration, theco-crystals or pharmaceutical compositions can be administered orally,intranasally, transdermally, intradermally, vaginally, intraaurally,intraocularly, buccally, rectally, transmucosally, or via inhalation, orintravenous administration. The compositions may be deliveredintravenously via a balloon catheter. The compositions can beadministered to an animal (e.g., a mammal such as a human, non-humanprimate, horse, dog, cow, pig, sheep, goat, cat, mouse, rat, guinea pig,rabbit, hamster, gerbil, ferret, lizard, reptile, or bird).

The co-crystals or pharmaceutical compositions of this invention alsocan be delivered by implantation (e.g., surgically) such with animplantable device. Examples of implantable devices include, but are notlimited to, stents, delivery pumps, vascular filters, and implantablecontrol release compositions. Any implantable device can be used todeliver the compound VX-950 as the active ingredient in the co-crystalsor pharmaceutical compositions of this invention, provided that 1) thedevice, compound VX-950 and any pharmaceutical composition including thecompound are biocompatible, and 2) that the device can deliver orrelease an effective amount of the compound to confer a therapeuticeffect on the treated patient.

Delivery of therapeutic agents via stents, delivery pumps (e.g.,mini-osmotic pumps), and other implantable devices is known in the art.See, e.g., “Recent Developments in Coated Stents” by Hofma et al.,published in Current Interventional Cardiology Reports, 2001, 3: 28-36,the entire contents of which, including references cited therein, areincorporated herein. Other descriptions of implantable devices, such asstents, can be found in U.S. Pat. Nos. 6,569,195 and 6,322,847, and PCTInternational Publication Numbers WO 2004/044405, WO 2004/018228, WO2003/229390, WO 2003/228346, WO 2003/225450, WO 2003/216699, WO03/0204168, WO 2008/098255 A2, WO 2008/027872 A2, WO 2008/027871 A2, WO2007/140320 A2, WO 2006/124823A2, WO 2007/128969 A2, WO 2007/030478 A2,and WO 2005/120393 A2, each of which (as well as other publicationscited herein) is incorporated herein in its entirety.

Described below are examples of preparing and characterizing co-crystalsof this invention, which are meant to be only illustrative and not to belimiting in any way.

Example 1 Preparation by Ball-Milling Method

VX-950 and an equal molar equivalent of a co-crystal former (e.g.,2,4-dihydroxybenzoic acid or 3-methoxy-4-hydroxybenzoic acid) can bemixed with a solvent (e.g., methyl ethyl ketone or ethyl acetate). Thecomponents can then be milled using a Wig-L-Bug apparatus, e.g., RetschMM200 (GlenMills Inc, Clifton, N.J.) for 10 minutes at the frequency of15 Hz. After milling, a batch is dried, e.g., in a vacuum oven at 75° C.for 2 hours, to give a co-crystal of the invention.

Example 2 Preparation by Melting Method

VX-950 and an equal molar equivalent of a co-crystal former (e.g.,2,4-dihydroxybenzoic acid or 3-methoxy-4-hydroxybenzoic acid) can bemixed, e.g., by vortex for 5 minutes, with or without a solvent. Themixture is then placed in a reaction block (e.g., RR 98072 from RadleyDiscovery Technologies) with the lid closed and heated to the endotherm.The mixture is held for 30 minutes at the endotherm temperature, andthen the resulting mixture was cooled off under ambient conditions withthe lid off, and the solvent, when used, removed to give a co-crystal ofthe invention.

Example 3 Preparation by Solvent-Evaporation Method

2,4-Dihydroxybenzoic acid: 200 mg of VX-950 and 80 mg of2,4-dihydroxybenzoic acid (Sigma Chemicals Co., St. Louis, Mo., USA)were charged into a 20 mL glass vial. To the vial were then added 100 μLof dichloromethane and 100 μL of acetonitrile. The vial containing themixture was capped and the mixture was stirred at the room temperaturewith a magnetic stir bar at 600 rpm for 16 hours. The crystalline solidwas isolated and the liquid on its surface was removed by filter paperto give a co-crystal of VX-950 and 2,4-dihydroxybenzoic acid.

3-Methoxy-4-hydroxybenzoic acid: 200 mg of VX-950 and 80 mg of3-methoxy-4-hydroxybenzoic acid (Sigma Chemicals Co., St. Louis, Mo.,USA) were charged into a 20 mL glass vial. To the vial were then added100 μL of dichloromethane and 100 μL of acetonitrile. The vialcontaining the mixture was capped and the mixture was stirred at theroom temperature with a magnetic stir bar at 600 rpm for 72 hours. Thecrystalline solid was isolated and the liquid on its surface was removedby filter paper to give a co-crystal of VX-950 and3-methoxy-4-hydroxybenzoic acid.

2,5-Dihydroxbenzoic acid: 200 mg of VX-950 and 80 mg of2,5-dihydroxybenzoic acid (Sigma Chemicals Co., St. Louis, Mo., USA)were charged into a 20 mL glass vial. To the vial were then added 100 μLof dichloromethane and 100 μL of acetonitrile. The vial containing themixture was capped and the mixture was stirred at the room temperaturewith a magnetic stir bar at 600 rpm for 16 hours. The crystalline solidwas isolated and the liquid on its surface was removed by filter paperto give a co-crystal of VX-950 and 2,5-dihydroxybenzoic acid.

Example 4 Single Crystal Diffraction

Single crystal diffraction of the co-crystals can be performed on aBruker APEX II CCD diffractometer at 100K using Cu Kα radiation by usingsingle crystals picked from mother liquors and mounted on glass fibers.The crystals are cooled to 100K in a nitrogen flow system andoscillation photos were taken around ω axis at 4 φ angles. The data areindexed, integrated, and scaled with the APEX software. The structurescan be solved and refined with the SHELX-TL package.

Example 5 Thermogravimetric Analysis (TGA)

TGA of each sample was performed using a Model Q500 ThermogravimetricAnalyzer (TA Instruments, New Castle, Del., USA), which uses its controlThermal Advantage Q Series™ software, Version 2.2.0.248, ThermalAdvantage Release 4.2.1 (TA Instruments-Water LLC), with the followingcomponents: QAdv.exe version 2.2 build 248.0; RhDII.dII version 2.2build 248.0; RhBase.dII version 2.2 build 248.0; RhComm.dII version 2.2build 248.0; TaLicense.dII version 2.2 build 248.0; and TGA.dII version2.2 build 248.0. In addition, the analysis software used was UniversalAnalysis 2000 software for Windows 2000/XP, version 4.1 D build 4.1.0.16(TA Instruments).

For all of the experiments, the basic procedure for performing TGAincluded transferring an aliquot (about 3-8 mg) of a sample into aplatinum sample pan (Pan: Part No. 952018.906, TA Instruments). The panwas placed on a loading platform and was then automatically loaded intothe Q500 Thermogravimetric Analyzer using the control software.Thermograms were obtained by individually heating the sample at 10°C./minute across a temperature range (generally from the roomtemperature to 400° C. under flowing dry nitrogen (compressed nitrogen,grade 4.8 (BOC Gases, Murray Hill, N.J., USA), with a sample purge flowrate of 90 L/minute and a balance purge flow rate of 10 L/minute.Thermal transitions (e.g. weight changes) were viewed and analyzed usingthe analysis software provided with the instrument.

As in FIG. 2, the TGA spectrum of the co-crystal of VX-950 and2,5-dihydroxybenzoic acid (molar ratio being 1) showed approximateshowed continuous weight loss from approximately 210° C.

As in FIG. 5, the TGA spectrum of the co-crystal of VX-950 and3-methoxy-4-hydroxybenzoic acid (molar ratio also being 1) showedcontinuous weight loss from approximately 155° C.

As in FIG. 8, the TGA spectrum of the co-crystal of VX-950 and2,4-dihydroxybenzoic acid showed continuous weight loss fromapproximately 165° C.

Example 6 Differential Scanning Calorimetry (DSC)

DSC analysis was performed using an MDSC Q100 Differential ScanningCalorimeter (TA Instruments), which uses its control Thermal Advantage QSeries™ software, version 2.2.0.248, Thermal Advantage Release 4.2.1,with the following components: QAdv.exe version 2.2 build 248.0;RhDII.dII version 2.2 build 248.0; RhBase.dII version 2.2 build 248.0;RhComm.dII version 2.2 build 248.0; TaLicense.dII version 2.2 build248.0; and DSC.dII version 2.2 build 248.0. In addition, the analysissoftware used was Universal Analysis 2000 software for Windows 2000/XP,version 4.1 D build 4.1.0.16 (TA Instruments). The instrument wascalibrated with indium.

For all DSC analysis, an aliquot of a sample (approximately 2 mg) wasweighed into an aluminum sample pan (Pan: Part No. 900786.901; and Lid:Part No. 900779.901, TA Instruments). The sample pan was closed bycrimping with a single pinhole, allowed to equilibrate at 30° C., andthen loaded into the Q100 Differential Scanning Calorimeter which wasequipped with an autosampler. A thermogram was obtained by individuallyheating each sample at a rate at 50° C./minute across a temperaturerange (generally from the room temperature to 400° C.) under flowing drynitrogen (compressed nitrogen, grade 4.8 (BOC Gases, Murray Hill, N.J.,USA), with a sample purge flow rate of 60 L/minute and a balance purgeflow rate of 40 L/minute. An empty aluminum pan prepared the same way asthe pan with the sample was used a reference. Thermal transitions wereviewed and analyzed using the analysis software provided with theinstrument.

As in FIG. 3, the DSC thermogram shows the co-crystal of VX-950 and2,5-dihydroxybenzoic acid (molar ratio being 1:1) melt at approximately163.48° C.

As in FIG. 6, the DSC thermogram shows the co-crystal of VX-950 and3-methoxy-4-hydroxybenzoic acid (molar ratio being 1:1) melt atapproximately 178.09° C.

As in FIG. 9, the DSC thermogram shows the co-crystal of VX-950 and2,4-dihydroxybenzoic acid (molar ratio being 1:1) as an acetonitrilesolvate melt at about 150.81° C.

Example 7 X-ray Powder Diffraction (XRPD)

The XRPD pattern was obtained at the room temperature in reflection modeby using a Bruker D8 Discover diffractometer that was equipped with asealed tube source and a Hi-Star area detector (Bruker AXS, Madison,Wis., USA). A copper target X-ray tube (Siemens) was operated at 40 kVand 35 mA. Graphite monochromator and 0.5 mm collimator provided byBruker were used to produce parallel, monochromatic beam (CuKa, λ=1.5418Å). The distance between the sample and the detector was approximately30 cm. The sample was placed on a Si zero-background wafer (The GemDugout, State College, Pa.) which was then positioned and centered onXYZ platform. Data were acquired using GADDS software for Windows NT,version 4.1.16 (Bruker AXS, Madison, Wis., USA). Two frames wereregistered with an exposure time of 120 seconds per frame each at 2different 2θ angles: 8° and 26°. The sample was oscillated in both X andY directions with an amplitude of 1 mm during the exposure. The datawere subsequently integrated over the range of 3° to 41° 2-Theta with astep size of 0.02° and merged into one continuous pattern. Corundumplate (NIST standard 1976) was used to calibrate the instrument.

As shown in FIG. 1, the XRPD pattern of the co-crystal of VX-950 and2,5-dihydroxybenzoic acid (molar ratio being 1:1) showed peaks at about8.292, 9.614, 11.675, 12.488, 12.897, 13.120, 14.649, 17.078, 17.514,18.235, 19.241, and 20.323 degree 2-Theta.

As shown in FIG. 4, the XRPD pattern of the co-crystal of VX-950 and3-methoxy-4-hydroxybenzoic acid (molar ratio being 1:1) showed peaks atabout 7.788, 8.640, 9.376, 9.917, 11.410, 11.943, 12.749, 13.166,14.780, 16.512, 16.909, 17.734, 18.145, 18.823, 19.761, 20.674, 21.702,22.887, 23.372, 24.042, and 24.863 degree 2-Theta.

As shown in FIG. 8, the XRPD pattern of the co-crystal of VX-950 and2,4-dihydroxybenzoic acid (molar ratio being 1:1) showed peaks at about7.581, 8.532, 9.622, 11.859, 12.920, 14.815, 17.291, 17.827, 18.905, and20.588 degree 2-Theta.

Example 8 Solubility Analyses

An aliquot of a co-crystal of this invention can be placed in a tube andthen an aqueous medium is added. At set time points, an aliquot ofsupernatant is withdrawn, filtered through 0.45 PTFE micron filter(Millex, LCR, Millipore) and processed for high performance liquidchromatography (HPLC) analysis (Agilent 1100; Palo Alto, Calif., USA).The system is equipped with an autosampler set at 25° C. For the samplehandling, an aliquot of the co-crystal can be diluted with acetonitrileat 1 to 1 by v/v ratio. The samples can be run isocratically with thedetector set at 270 nm with a column being XTerra® Phenyl column 150mm×4.6 mm, 3.5 μm Particle Size (P/N 186001144) (Waters, Milford, Mass.,USA). The mobile phase can be potassium phosphate buffer (10 mM,pH=7.0):methanol at 60:40 (v/v) ratio. The run can be done at theflow-rate of 1 mL/minute and completed within 15 minutes.

The water solubility data can be determined at ambient conditions byequilibrating the co-crystal with water on a shaking bed for 24 hoursfollowed by centrifugation and separation of the saturated solution. Thesolubility in simulated gastric and intestinal fluids (both fed andfasted) can be determined at room temperature by adding the co-crystalto the simulated fluid under continuous stirring for 24 hours. Atselected time points, samples are filtered and the filtrate assayed byHPLC.

Example 9 Suspension Stability

The physical stability of a co-crystal of this invention upon suspensionin aqueous media can also evaluated. Specifically, the co-crystal powdercan be slurried, e.g., in (1) unbuffered, deionized water and (2) a 1%(w/w) solution of HPMC (low viscosity grade) at 25° C. at a nominalconcentration of approximately 6 mg/mL. Slurries can then mixed using amagnetic stir bar and plate. The samples of the solid can be isolated byfiltration, e.g., at time intervals of 1, 2, 6 and 24 hours.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A co-crystal comprising VX-950 and 2,5-dihydroxybenzoic acid.
 2. Theco-crystal of claim 1, wherein the molar ratio of VX-950 and2,5-dihydroxybenzoic acid is in the range of about 5:1 to about 1:5. 3.The co-crystal of claim 1, wherein the molar ratio of VX-950 and2,5-dihydroxybenzoic acid is about 1:1.
 4. The co-crystal of claim 3,having at least two of the four X-ray powder diffraction peaks at about8.9, 9.6, 11.7, and 17.1 degree 2-Theta, each with a standard deviationof about +/−0.3 degree 2-Theta.
 5. The co-crystal of claim 3, having apeak in its DSC thermogram at about 163.48° C. with a standard deviationof about +/−5° C.
 6. A co-crystal comprising VX-950 and2,4-dihydroxybenzoic acid.
 7. The co-crystal of claim 6, wherein themolar ratio of VX-950 and 2,4-dihydroxybenzoic acid is in the range ofabout 5:1 to about 1:5.
 8. The co-crystal of claim 6, wherein the molarratio of VX-950 and 2,4-dihydroxybenzoic acid is about 1:1.
 9. Theco-crystal of claim 8, having at least two of the four X-ray powderdiffraction peaks at about 7.6, 8.5, 9.6, and 11.9 degree 2-Theta, eachwith a standard deviation of about +/−0.3 degree 2-Theta.
 10. Theco-crystal of claim 8, having a peak in its DSC thermogram at about150.81° C. with a standard deviation of about +/−5° C.
 11. A co-crystalcomprising VX-950 and 3-methoxy-4-hydroxybenzoic acid.
 12. Theco-crystal of claim 11, wherein the molar ratio of VX-950 and3-methoxy-4-hydroxybenzoic acid is in the range of about 5:1 to about1:5.
 13. The co-crystal of claim 11, wherein the molar ratio of VX-950and 3-methoxy-4-hydroxybenzoic acid is about 1:1.
 14. The co-crystal ofclaim 13, having at least two of the four X-ray powder diffraction peaksat about 7.8, 8.6, 11.4, and 11.9 degree 2-Theta, each with a standarddeviation of about +/−0.3 degree 2-Theta.
 15. The co-crystal of claim13, having a peak in its DSC thermogram at about 178.09° C. with astandard deviation of about +/−5° C.
 16. A co-crystal comprising VX-950;a co-crystal former selected from the group consisting of3-methoxy-4-hydroxybenzoic acid, 2,4-dihydroxybenzoic acid, and2,5-dihydroxybenzoic acid; and a solvent selected from the groupconsisting of dichloromethane, acetonitrile, ethyl acetate, ethanol,acetone, and mixtures thereof.
 17. The co-crystal of claim 16, whereinthe solvent is acetonitrile, chloromethane, or their mixture.
 18. Theco-crystal of claim 16, wherein the co-crystal former is3-methoxy-4-hydroxybenzoic acid.
 19. The co-crystal of claim 18, whereinthe molar ratio of VX-950 and 3-methoxy-4-hydroxybenzoic acid is in therange of about 5:1 to about 1:5.
 20. The co-crystal of claim 18, whereinthe molar ratio of VX-950 and 3-methoxy-4-hydroxybenzoic acid is about1:1.
 21. The co-crystal of claim 20, having at least two of the fourX-ray powder diffraction peaks at about 7.788, 8.640, 11.410, and 11.943degree 2-Theta, each with a standard deviation of about +/−0.3 degree2-Theta.
 22. The co-crystal of claim 20, having a DSC peak in its DSCthermogram at about 178.09° C. with a standard deviation of about +/−5°C.
 23. The co-crystal of claim 20, wherein the solvent is a mixture ofacetonitrile and dichloromethane.
 24. The co-crystal of claim 16,wherein the co-crystal former is 2,4-dihydroxybenzoic acid.
 25. Theco-crystal of claim 24, wherein the molar ratio of VX-950 and2,4-dihydroxybenzoic acid is in the range of about 5:1 to about 1:5. 26.The co-crystal of claim 24, wherein the molar ratio of VX-950 and2,4-dihydroxybenzoic acid is about 1:1.
 27. The co-crystal of claim 26,having at least two of the four X-ray powder diffraction peaks at about7.581, 8.532, 9.622, and 11.859 degree 2-Theta, each with a standarddeviation of about +/−0.3 degree 2-Theta.
 28. The co-crystal of claim26, having a DSC peak in its DSC thermogram at about 150.81° C. with astandard deviation of about +/−5° C.
 29. The co-crystal of claim 26,wherein the solvent is a mixture of acetonitrile and dichloromethane.30. The co-crystal of claim 16, wherein the co-crystal former is2,5-dihydroxybenzoic acid.
 31. The co-crystal of claim 30, wherein themolar ratio of VX-950 and 2,5-dihydroxybenzoic acid is in the range ofabout 5:1 to about 1:5.
 32. The co-crystal of claim 30, wherein themolar ratio of VX-950 and 2,5-dihydroxybenzoic acid is about 1:1. 33.The co-crystal of claim 32, having at least two of the four X-ray powderdiffraction peaks at about 8.292, 9.614, 11.675, and 17.078 degree2-Theta, each with a standard deviation of about +/−0.3° 2-Theta. 34.The co-crystal of claim 32, having a DSC peak in its DSC thermogram atabout 163.48° C. with a standard deviation of about +/−5° C.
 35. Theco-crystal of claim 32, wherein the solvent is a mixture of acetonitrileand dichloromethane.
 36. A pharmaceutical composition comprising aVX-950 co-crystal, which comprises VX-950 and a co-crystal formerselected from the group consisting of 3-methoxy-4-hydroxybenzoic acid,2,4-dihydroxybenzoic acid, and 2,5-dihydroxybenzoic acid.
 37. Thepharmaceutical composition of claim 36, wherein the molar ratio ofVX-950 and the co-crystal former is in the range of about 5:1 to about1:5.
 38. The pharmaceutical composition of claim 36, wherein the molarratio of VX-950 and the co-crystal former is about 1:1.
 39. Thepharmaceutical composition of claim 36, further comprising apharmaceutically acceptable diluent, solvent, excipient, carrier, orsolubilizing agent.
 40. A method of making a co-crystal of claim 16,comprising: a. providing VX-950, b. providing a co-crystal formerselected from the group consisting of 3-methoxy-4-hydroxybenzoic acid,2,4-dihydroxybenzoic acid, and 2,5-dihydroxybenzoic acid, c. grinding,heating, co-subliming, co-melting, or contacting in solution VX-950 withthe co-crystal former under crystallization condition so as to form theco-crystal in solid phase, and d. optionally isolating the co-crystalformed by step (c).
 41. A method for modulating a chemical or physicalproperty of interest of a co-crystal of claim 16, comprising: a.measuring the chemical or physical property of interest for VX-950 and aco-crystal former selected from the group consisting of3-methoxy-4-hydroxybenzoic acid, 2,4-dihydroxybenzoic acid, and2,5-dihydroxybenzoic acid, b. determining the mole fraction of theVX-950 and the co-crystal former that will result in the desiredmodulation of the chemical or physical property of interest, and c.preparing the co-crystal with the molar fraction determined in step (b).